Characterization of toxicity determinants in Bacillus thuringiensis mosquitocidal delta-endotoxins.

摘要

Bacillus thuringiensis mosquitocidal toxin Cry4Ba has no significant natural activity against Culex quinquefasciatus or Cx. pipiens (LC50 > 80,000 ng/ml and LC50 > 20,000 ng/ml, respectively). We introduced into Cry4Ba amino acid substitutions to mimic the loop residues of domain II of Cry4Aa and created an engineered Cry4Ba toxin with high Culex toxicity (LC50 = 365 ng/ml and LC50 = 95 ng/ml for Cx. quinquefasciatus and Cx. pipiens , respectively). The mutant proteins were tested on 4 different species of important human disease vectors, Aedes aegypti, Anopheles quadrimaculatus, Cx. quinquefasciatus and Cx. pipiens. Loops 1 and 2 exchanges eliminated activity towards Ae. aegypti and An. quadrimaculatus but did not result in increased activity against Culex. Further mutations in loop 3 resulted in an increase in toxicity by >700-fold and >285-fold against Cx. quinquefasciatus (LC50 = 114 ng/ml) and Cx. pipiens (LC50 = 70 ng/ml), respectively, while having very little effect on the activity against the other two mosquitoes. These results suggest that by introducing short variable sequences of the loop regions from one toxin to another might provide a rational design approach of enhancing B. thuringiensis Cry toxins. A mutant of Cry19Aa in which the loops 1 and 2 mimicked Cry4Ba was enhanced in activity towards Ae. aegypti by 42,000-fold, but did not affect Anopheles and Culex activities of the wild type toxin. The significant increase in toxicity was not correlated with reversible or irreversible binding. These results suggest a complex mechanism of action of these mosquitocidal toxins. Aminopeptidase N (APN) type proteins isolated from several species of lepidopteran insects have been implicated as Bacillus thuringiensis (Bt) toxin-binding proteins (receptors). A 100-kDa protein with APN activity (APN-100) was isolated from the brush border membrane of Anopheles quadrimaculatus. Native state binding analysis by surface plasmon resonance shows that APN-100 forms tight binding with a mosquitocidal Bt toxin, Cry11Ba. The N-terminal sequence of APN-100 shares high homology with three putative aminopeptidase proteins from An. gambiae, suggesting that they might each be a candidate for Cry11Ba receptor.